The goal of this new R01 application is to investigate the mechanisms of T cell activation and the consequences in the progression of cardiac fibrosis (CF) in the deadly syndrome of heart failure (HF), currently the leading cause of mortality and hospitalizations in the USA. We were the first to report cardiac T cell infiltration associated with CF in patients with non-ischemic HF, and using the well- established experimental model of HF induced by transverse aortic constriction (TAC), we and others described a major role for CD4+ T cells as major contributors to non-ischemic HF. However, due to the complexity of the mechanisms of T cell activation resulting in inflammation, CF and HF, no immunomodulatory or anti-fibrotic therapies have yet translated to clinical practice to treat HF. Our preliminary data reveal the novel finding that T cell activation occurs in the mediastinal lymph nodes that drain the heart (mLN), and also within the heart in a classic dependent manner that involves T cell receptor (TCR) engagement by antigens presented by Major histocompatibility complex II (MHC-II) expressed on antigen presenting cells (APC). We additionally demonstrate that activated cardiac fibroblasts (CFB) express MHC-II and thus could function as APC in the heart. Emerging evidence suggests that T cells can also be activated by alarmins, soluble inflammatory mediators produced in response to sterile inflammation, through TCR independent pathways in a ?non classic TCR- independent manner, and our in vitro and in vivo preliminary data is in support of this. Based on these findings, we will test the central hypothesis that classic and non-classic T cell activation mechanisms cooperate to initiate and sustain CF during the progression of HF. In aim 1, we will use Nur77GFP mice, in which T cells express GFP only when stimulated classically through the TCR, to map the specific location and timing of classic T cell activation during TAC. We will additionally perform single cell TCR sequencing on heart sorted GFP+CD4+ T cells to identify the immunodominant T cell clones in HF progression. The APC responsible for such activation and its effects in CF and HF over time will be evaluated in cell specific MHC-II-/- mice. In aim 2, we will perform adoptive transfer experiments of WT and alarmin sensing-impaired activated CD4+ T cells into MHC-II-/- recipient mice, which lack classic T cell activation and are normally protected from CF and HF. CF, cardiac function and the alarmins responsible for T cell activation will be characterized in in vivo and in vitro assays. In aim 3, based on our data indicating that activated T cell adhesion to CFB induces their transformation to pro-fibrotic myofibroblast in a TGF? dependent manner, we will investigate the mechanisms of TGF? synthesis, release and signaling in CFB in response to classically and non classically activated T cell adhesion. We will use in vitro biochemical and molecular assays. These important studies will result in a deeper understanding of how best to regulate T cell activation and T cell induced in HF and provide new perspectives on how to prevent, ameliorate and treat non-ischemic HF.